Date of Award


Degree Name

Doctor of Philosophy



First Advisor

Dr. Andre R. Venter

Second Advisor

Dr. John B. Miller

Third Advisor

Dr. Yirong Mo

Fourth Advisor

Dr. Blair R. Szymczyna


Mass spectrometry, protein, ionization, electrospray ionization, desorption electrospray ionization


Mass spectrometry (MS) is an analytical technique in which a sample is converted to gas-phase ions that are subsequently separated and detected. It offers great speed, selectivity, and sensitivity during analysis, characteristics which have enabled it to become a leading method for the study of proteins. The applications of MS for these biologically significant macromolecules range from accurately determining identity and sequence to shedding light on post-translational modifications and protein-molecule interactions. As a first step towards analysis by MS, gas-phase protein ions must be formed. A common method for ionization is electrospray ionization (ESI), where a liquid sample including the protein is charged, nebulized, and evaporated, resulting in bare protein ions. Although ESI has been used in this way for over two decades, many aspects of the protein charging mechanism remain unclear.

To address this problem, my research has focused on (1) identifying the factors that determine the extent of protein multiple charging during ESI and (2) improving the ionization of proteins by desorption electrospray ionization (DESI). DESI is a method similar to ESI, except that the sample is desorbed from a surface by the spray instead of being present in it from the onset. As a result of (1), a simple model was developed that enables the accurate prediction of protein multiple charging observed during ESI-MS if the protein sequence is known. Furthermore, the enhancement of multiple charging that is observed upon the addition of certain organic reagents, a phenomenon known as supercharging, was investigated and a novel mechanism of protein supercharging was proposed. For (2), the difficulty in analyzing large proteins by DESI-MS was studied using an innovative approach where DESI was separated into its individual sub-processes and their individual contributions to the DESI process were evaluated. As a result, core limitations to the DESI-MS of large proteins were identified. The results of my cumulative research efforts should lead to the improved MS analysis of proteins by spray ionization methods, including ESI and DESI.


Dr. Bruce Bejcek was the 5th advisor.

Access Setting

Dissertation-Open Access